Compounds and Methods Useful for Detection and Treatment of Cancer

ABSTRACT

The present invention relates to compounds and methods useful for the detection and treatment of disorders associated with frameshift mutations in coding microsatellite regions. The compounds and methods are applicable in cancers, especially of DNA mismatch repair deficient (MMR) sporadic tumors and HNPCC associated tumors. The compounds are useful for detection of disorders and in therapy such as immuno-therapy. The diagnostic methods relate to diagnosis and prognostic assessment of disorders associated with frameshift polypeptides originating from frameshift mutations in coding microsatellite regions of genes based on the detection of immunological entities directed against said frameshift polypeptides in body fluids. With respect to the treatment of cancer, the invention pertains to methods which use immuno therapy with combinatorial mixtures of tumor specific frameshift peptides to elicit a cytotoxic T-cell response specifically directed against tumor cells for prevention and curative treatment of cancers and pre-cancers.

This is a continuation of U.S. Ser. No. 16/120,981, filed Sep. 4, 2018;which is a continuation of U.S. Ser. No. 14/340,467, filed Jul. 24,2014; which is a continuation of U.S. application Ser. No. 13/284,660,filed Oct. 28, 2011, now abandoned; which is a divisional of U.S. patentapplication Ser. No. 10/511,698, filed Jun. 2, 2005, U.S. Pat. No.8,053,552; which is a National Stage of International ApplicationPCT/EP03/04083, filed Apr. 17, 2003, published Oct. 23, 2003, under PCTArticle 21(2) in English; which claims the priority of EP 02008773.0,filed Apr. 18, 2002, EP 02008771.4, filed Apr. 18, 2002, and EP02008774.8, filed Apr. 18, 2002. The contents of the above-identifiedapplications are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to compounds and methods useful for thedetection and treatment of disorders associated with frameshiftmutations in coding microsatellite regions. The compounds and methodsare applicable in cancers, especially of DNA mismatch repair deficient(MMR) sporadic tumors and HNPCC associated tumors. The compoundsdisclosed in the invention are useful for detection of disorders and intherapy such as e.g. immuno-therapy. The diagnostic methods relate todiagnosis and prognostic assessment of disorders associated withframeshift polypeptides originating from frameshift mutations in codingmicrosatellite regions of genes based on the detection of immunologicalentities directed against said frameshift polypeptides in body fluids.With respect to the treatment of cancer, especially of DNA mismatchrepair deficient (MMR) sporadic tumors and HNPCC associated tumors, theinvention pertains to methods which use immuno therapy withcombinatorial mixtures of tumor specific frameshift peptides to elicit acytotoxic T-cell response specifically directed against tumor cells foruse in prevention as well as in curative treatment of cancers andpre-cancers.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The Sequence Listing is concurrently submitted herewith with thespecification as an ASCII formatted text file via EFS-Web with a filename of P31038US4_SEQLIST_ST25 with a creation date of May 10, 2021, anda size of 269,736 bytes. The Sequence Listing filed via EFS-Web is partof the specification and is hereby incorporated in its entirety byreference herein.

BACKGROUND OF THE INVENTION

Tumor cells accumulate mutations in components of cellular pathways,that are essential for the maintenance of normal growth anddifferentiation. In human epithelial tumors, 2 types of geneticinstability have been identified: chromosomal instability (CIN), whichmarks structural and numerical chromosomal aberration in aneuploidneoplastic cells, and microsatellite instability (MSI), which reflectslength variations at repetitive DNA sequences in diploid tumor cells.The type and spectrum of mutated genes markedly differs among CIN andMSI tumors, suggesting distinct but not mutually exclusive pathways ofcarcinogenesis. MSI occurs in about 90% of hereditary nonpolyposiscolorectal cancers (HNPCC) as well as in about 15% of sporadic tumors ofthe colon and other organs, and is caused by mutational inactivation ofdifferent DNA mismatch repair genes.

The mutations lead especially in the case of frameshift mutations incoding microsatellite regions to the expression of new peptide sequencesin the affected cells, that do not occur in wild-type cells. The alteredpeptides may be used as detection markers for disorders associated withframeshift mutations in coding microsatellite regions such asdegenerative disorders or cancers (e.g. gastrointestinal cancers).

The accumulation of genetic alterations and resulting mutant proteinsrepresent a major obstacle for tumor cells to escape immunesurveillance. The mutant proteins or peptides encoded by expressedmutant genes may elicit a specific cellular immune response and thus maybe recognized by CTL. This is especially true concerning mutationsresulting from chromosomal instabilities, but also pertains to moresubtle genetic alterations, such as small deletions and insertions inmicrosatellites.

This situation can be used for prevention and therapies in cancers. Thecancer cells are characterized by the expression of neo-peptides, whicharouse from the genetic alterations. These proteins are not present innormal, non cancerous tissues. Thus, the neo-peptides may be used todistinguish on a molecular level between tumors and normal tissues. Astools suitable for the molecular discrimination antigen specificmolecules or cells may be used. Thus it is possible to elicit an immuneresponse against frameshift peptides, as might arise from MSI mutations,specific for tumors. Using cytotoxic T lymphocytes or FS8-specificantibodies it is thus possible to specifically attack and eliminatetumor cells in organisms and tissues.

A large number of genes containing coding microsatellites have beenidentified. Various of these genes show mutations within themicrosatellites with certain frequencies in sporadic tumors. For few ofthese genes it could be shown, that they are involved in the majority ofcases of particular tumors. For example the (A)₁₀ tract within the TGWUgene and the (G)₈ tract within the BAX gene are commonly mutated ingastrointestinal cancers such as colon cancer or gastric cancer.

The T-cell mediated immune response on the other hand provides a strongand specific selection pressure for the rapidly growing tumor cells.(Tomlinson I, et al., Nat. Med. 1999; 5: 11-2) Thus tumor evolution isforced to circumvent the immune surveillance by the cellular immuneresponse. As a consequence of this selection pressure, mutations ingenes of the antigen processing or presenting machinery arise inMMR-deficient tumor cells. In fact evasion of the immune surveillance byacquiring β2-microglobulin mutations has been observed at high frequencyin MSI⁺ tumor cells (Bicknell D C, et al., Curr. Biol 1996; 6: 1695-7).Other targets of specific mutation or down-regulation of expression areTAP1/TAP2 or HLA alleles. Direct down-regulation of expression ofimmunogenic epitopes is also a possible mechanism of immune escape ashas been shown for melanoma-associated antigens in vivo (Jager E, etal., Int. J. Cancer 1997; 71: 142-7). Due to this fact the relevantepitopes may not be detectable by the immune system.

As a consequence the promising therapies based on immune responsedirected against frameshift peptides in tumor tissues may fail in a notyet determined number of cases of tumors. So the approaches for the useof the vaccination therapy of cancer mediated by frameshift peptidesmight be limited by the fact, that the potential immunogenic frameshiftpeptides are not always detectable by the immune system due to mutationsin the antigen presenting and processing properties of the cancer cells.

Even supposed, that during specific stages of tumor development cellsexpress the aberrant proteins and thus are vulnerable to the immunesurveillance, there potentially remains a population of tumor cells,that is not eliminated by the therapy. This state is quite undesirablefor any therapy of cancers, for the remaining cells may continue growingand thus the tumor is not eliminated from the organism.

Similarly methods for detection of molecular markers suitable fordiagnosis of disorders associated with frameshift mutations in codingmicrosatellite regions and for assessment of prognosis for saiddisorders are prone to overlook especially markers originating fromsmall populations of affected cells as they may occur particularly inearly stages of the disorder. This may in part be overcome by anelevated effort in the preparation of testing samples to raise theprobability of the detection of disorders. However especially concerningdisorders located in body regions, that are merely inaccessible, oraccessible only under circumstances, that are quite consuming ordiscomfortable to patients alternative methods for reliable detection ofmolecular markers associated with disorders are desirable.

It is known in the art, that the mutant proteins or peptides derivedfrom aberrantly expressed proteins may elicit a specific cellular immuneresponse and thus may be recognized by cytotoxic T lymphocytes (CTL).This is especially true concerning mutations resulting from chromosomalinstabilities, but pertains also to the more subtle genetic alterations,such as small deletions and insertions in microsatellites.

The T-cell mediated immune response on the other hand provides a strongand specific selection pressure for the rapidly growing tumor cells(Tomlinson I, et al., Nat. Med. 1999; 5: 11-2). Thus tumor evolution isforced to circumvent the immune surveillance by the cellular immuneresponse. As a consequence of this selection pressure, mutations ingenes of the antigen processing or presenting machinery arise in DNAmismatch repair (MMR)-deficient tumor cells. In fact evasion of theimmune surveillance by acquiring β2-microglobulin mutations has beenobserved at high frequency in MSI⁺ tumor cells (Bicknell D C, et al.,Curr. Biol 1996; 6: 1695-7). Other targets of specific mutation ordown-regulation of expression are TAP1/TAP2 or HLA alleles. Directdown-regulation of expression of immunogenic epitopes is also a possiblemechanism of immune escape as has been shown for melanoma-associatedantigens in vivo (Jager E, et al., Int. J. Cancer 1997; 71: 142-7). Dueto this fact the relevant epitopes may not be detectable by the immunesystem. Thus it was suspected, that components of the immune systemdirected against novel peptides, characteristic for e.g. tumor cells,are not suitable for the detection of the disorders especially in thecase of frameshift mutation peptides. This is due to the fact, that MSIdisorders are frequently associated with DNA mismatch repair deficiencyand thus are especially prone to mutations. This makes the affectedcells especially apt to escape mutations in response to the attack bythe immune system.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1:

ELISpot-analysis of FSP T-cell lines. Titrated amounts of T-cells wereincubated overnight with 3.5×10⁴ peptide loaded T2 cells per well. Thenumber of IFN-γ-releasing activated T-cells (spots) among the totalnumber of cells analyzed (10⁶) is depicted for each frameshift peptide.Reactivity against peptide YLLPAIVHI from the nuclear protein P68 servedas a negative control and is indicated (open bars).

FIGS. 2-1 to 2-8:

Listing of sequences of polypeptides encoded by genes with codingmicrosatellites; the sequences of polypeptides arising from differentpossible frameshift mutations are depicted. For each polypeptide thesequence expressed from the wild type open reading frames is given(wtORF); Furthermore the sequences expressed from (−1) mutations andfrom (−2/+1) mutation are given.

FIG. 3: Frameshift peptide specific and HLA-A2-restricted lysis oftarget cells. (FSP02); The antigen specificity of the FSP02 CTL line wastested in the presence of unlabeled cold targets, T2 cells pulsed withFSP02 (open squares) at an inhibitor:target ratio of 50:1. Lysis withoutcold targets is shown as a control (closed squares). All data are shownas the mean and standard deviation from 3 replicate wells. Forexperimental details see example 6.

FIG. 4: Frameshift peptide specific and HLA-A2-restricted lysis oftarget cells. (FSP06); The antigen specificity of the FSP06 CTL line wastested in the presence of unlabeled cold targets, T2 cells pulsed eitherwith FSP06 (open squares) at an inhibitor:target ratio of 50:1. Lysiswithout cold targets is shown as a control (closed squares). All dataare shown as the mean and standard deviation from 3 replicate wells. Forexperimental details see example 6.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have now surprisingly found, that specific antibodies orantigen recognizing cells directed against a particular new frameshiftpeptide are in detectable levels present within the body fluids ofindividuals harbouring MSI associated disorders. This is especially dueto the fact, that during specific stages of tumor development cellsexpress the aberrant proteins. During these stages the peptides may beaccessible to the immune surveillance, and thus an immune response maybe elicited. Even though cell populations affected by respectivedisorders such as tumors may be eliminated from the organism, or may bemutated, such, that no further presentation of immunogenic epitopes mayoccur, there remains an immunological memory of the presence of therespective peptides at a certain time. Such the evolution of a cellpopulation affected by a disorder associated with the expression of newframeshift peptides leaves immunological traces of its existenceconsisting e.g. of antibodies and specific T-lymphocytes directedagainst the particular peptides. Hence the presence of specific immuneresponse elements directed against a particular frameshift peptide isindicative of the presence of a population of (tumor) cells, thatexpresses or has expressed at a certain time the respective peptide.

To further enhance the fidelity of the detection of the presence orabsence of a disorder a set of peptides frequently occurring indisorders such as tumors may be applied in the detection reaction.

The present invention thus provides a method for the detection ofdisorders associated with frameshift peptides arising from mutations incoding microsatellite regions based on the detection of specificimmunological entities directed against said frameshift peptides presentin the body fluids of affected individuals directed against saidframeshift peptides. The method is suited for primary detection ofdisorders such as tumors, for the early detection of disorders or ofprecursory stages of disorders, and for the assessment of prognosis insaid disorders.

One further aspect of the invention is based on the inventors findings,that a mixture of frameshift peptides chosen according to combinatorialparameters minimizes the escape of particular populations of tumor cellsfrom immunogenic elimination and may be used as a vaccine against a widevariety of DNA mismatch repair deficient tumors. The invention is basedon the fact, that the immune escape of tumor cells is directed towardssingle immunogenic epitopes (frameshift peptides) in a potentiallyrestricted subpopulation of the tumor cells. In the vast majority ofMSI+ tumors various mutations may be found within the genome of theaffected cells. So a combinatorial vaccination approach includingseveral antigenic peptides could overcome this obstacle.

It is one aspect of the present invention to provide nucleic acidsequences of the genes TAF1B, MACS, UVRAG, ELAVL3, TCF6L1, ABCF1, AIM2,CHD2 and HT001, that have frameshift mutations within their codingregions.

A second aspect of the present invention is to provide new frameshiftpeptides, that occur in a wide range of different MSI⁺ tumors.

A third aspect of the present invention is a method for detection ofMSI⁺ tumors using said frameshift peptides as molecular markers.

A fourth aspect of the present invention is a method for treatment ofMSI⁺ tumors using said frameshift peptides for therapeutic purposes.

In a fifth aspect the present invention provides sets of frameshiftpeptides, that occur in a wide range of different MSI⁺ tumors and thatdiminish the probability of escape of tumors to be attacked by theimmuno-therapy.

A sixth aspect of the present invention is a method for vaccinationagainst MSI⁺ tumors using said set of frameshift peptides.

A seventh aspect of the present invention is a method for treatment ofMSI⁺ tumors using a set of frameshift peptides, that elicit an immuneresponse directed against a wide range of tumors.

An eighth aspect of the invention relates to the detection of disordersassociated with frameshift mutations in coding microsatellite regionscomprising the detection of immunological entities directed againstframeshift peptides in body fluids of individuals.

The present invention thus provides compounds and methods for thetherapy and detection of disorders associated with frameshift peptidesarising from mutations in coding microsatellite regions. The diagnosticand therapeutic methods are suited for application in disorders such astumors or in precursory stages of disorders.

Within the context of the present invention disorders associated withframeshift mutations comprises for example degenerative diseases, suchas neurodegenerative diseases, vascular disorders, disorders caused bystress, such as oxidative stress, chemically induced stress, irradiationinduced stress, etc. and cancers including all sporadic cancers as wellas HNPCC associated cancers. Cancers as used herein may comprise e.g.colorectal cancer, small cell lung cancer, liver cancer (primary andsecondary), renal cancer, melanoma, cancer of the brain, head and neckcancer, gastrointestinal cancers, leukemias, lymphomas. prostate cancer,breast cancer, ovary cancer, endometrial cancer, lung cancer, bladdercancer etc.

The method according to the present invention may be applied to anyeucaryotic organisms. In one aspect the eucaryotic organisms are thoseexhibiting an immunologic defense system. The eucaryotic organisms arefor example mammalian animals and especially animals of agriculturalvalue such as pigs, cows, sheep, etc., companion animals, such as cats,dogs, horses etc., animals employed in research purposes such as mice,rats, rabbits, hamsters etc. or humans.

Nucleic acid molecules according to the present invention may comprisepolynucleotides or fragments thereof. Preferred polynucleotides maycomprise at least 20 consecutive nucleotides, preferably at least 30consecutive nucleotides and more preferably at least 45 consecutivenucleotides of the sequences. The nucleic acids according to the presentinvention may also be complementary or reverse complementary to any ofsaid polynucleotides. Polynucleotides may for example includesingle-stranded (sense or antisense) or double-stranded molecules, andmay be DNA (genomic, cDNA or synthetic) or RNA. RNA molecules compriseas well hnRNA (containing introns) as mRNA (not containing introns).According to the present invention the polynucleotides may also belinked to any other molecules, such as support materials or detectionmarker molecules, and may, but need not, contain additional coding ornon-coding sequences.

Mutation as used in the context of the present invention may compriseinsertions or deletions of one or several nucleotides (or nucleotiderepeats) within the specified microsatellite sequences. In a preferredembodiment of the present invention the number of nucleotides to beinserted or deleted is not 3 or must not be divisible by 3, such thatthe mutation leads to a frameshift with respect to the translationalreading frame of the downstream nucleic acid sequence. Thus the nucleicacid sequence downstream of the mutation point will render apolypeptide-sequence different from the native sequence encoded by therespective gene. The mutation in these cases leads to a novel peptidesequence (a neo-peptide). Commonly the new peptide sequence is short dueto the fact, that novel stop codons arise from the shift in the readingframe.

Frameshift mutations in microsatellites are usually due to DNApolymerase slippage and may be characterized by the type of the repeat.Thus in mono nucleotides repeats this type of mutation renders 1 ntinsertion or deletion. In dinucleotide repeats and tetranucleotiderepeats mutations are insertions or deletions of 2 or 4 nt respectively.For example commonly (−1) mutations occur in mononucleotide repeats(MNRs). In these mutations one nucleotide is deleted such that thereading frame is shifted by one nucleotide toward the 5′ end of the genecompared to the original reading frame. This type of mutation renders areading frame identical to that produced by (+2) mutations, which arisefrom two nucleotide insertions in the respective microsatellites. Therespective (−1) or (+2) polypeptides might differ by one amino acid. Theother frameshift mutation variant leading to frameshift mutationsdiffering from (−1) mutations concerning the resulting reading frame isthe (+1) mutation, arising from one nucleotide insertion, thus renderinga reading frame one nucleotide toward the 3′ end of the gene compared tothe original reading frame. This mutation type gives a reading frameidentical to that of (−2) mutations, wherein the encoded polypeptidesdiffer by one amino acid. (−3) and (+3) mutations are irrelevantaccording to the present invention, for they do not give rise toframeshift polypeptides.

Frameshift polypeptides as used herein shall comprise any polypeptidesor fragments thereof, that arise by a frameshift mutation within acoding microsatellite sequence of a gene. A gene may harbour one or morecoding microsatellite regions, that may give rise to frameshiftpeptides. The coding microsatellites according to the present inventioncomprise mononucleotide repeats, dinucleotide repeats, trinucleotiderepeats, tetranucleotide repeats and pentanucleotide repeats of anylength. According to present invention coding microsatellites containpreferably at least 3 and more preferably at least 5 repeats of therespective nucleotide pattern (1-5 nucleotides, which are repeated).

The frameshift polypeptides as used according to the present inventioncomprise at least 1, more preferred at least 2 and even more preferredat least 3 amino acids of the mutated part of the polypeptide.Additionally, the frameshift polypeptides may comprise fragments of theoriginally non-mutated proteins and/or may be fused to any otherpolypeptide sequences suitable for the purposes of the presentinvention. Examples of such polypeptide sequences are linker sequences,or structural peptide sequences, such as beta barrels, loop sequencesetc. that facilitate the immunogenicity of the frameshift sequencesaccording to the present invention within the fusion polypeptide.

In certain embodiments of the invention the frameshift polypeptidessuitable for the methods disclosed herein are immunogenic polypeptides.This requires, that the polypeptides may stimulate immune responses inhost organisms either in the form the polypeptides adopt in theirnatural environment and/or especially in the form the polypeptides adoptafter processing by the cellular antigen processing and presentingmachinery.

According to the present invention the frameshift polypeptides may alsobe represented by nucleic acids coding for said polypeptides. Thesenucleic acids may for example be used for the in situ expression of therespective polypeptides. For the purpose of expression of nucleic acids,the particular nucleic acids may be joined with suitable other nucleicacid sequences, that enable for the cloning and expression of saidnucleic acids encoding the frameshift polypeptides.

In certain embodiments of the present invention frameshift polypeptidesmay comprise fusion or chimeric polypeptides containing sequencesdisclosed herein. Fusion proteins comprise the frameshift polypeptideaccording to the present invention together with any second and furtherpolypeptides, such as e.g. one more frameshift polypeptide of the samesequence or of another sequence. Heterologous polypeptides may compriseenzymes, receptor molecules, antigens, antigenic or immunogenic epitopesor fragments, antibodies or fragments thereof, signalling polypeptidesor signal transducing polypeptides, labelled polypeptides etc. In oneembodiment of the invention the fusion peptides may be constructed forenhanced detection or purification of the frameshift polypeptides, or ofcomplexes of the frameshift polypeptides with the respectiveimmunological entities according to the present invention. For thepurpose of purification tags, such as e.g. his-tags, myc-tags etc. maybe added to the polypeptides. For the purpose of detection antigenicportions, enzymes, chromogenic sequences etc. may be fused to thepolypeptides. The fusion proteins of the present invention may (but neednot) include a linker peptide between the first and second polypeptides.

A nucleic acid sequence encoding a fusion protein of the presentinvention is constructed using known recombinant DNA techniques toassemble separate nucleic acid sequences encoding the first and secondpolypeptides into an appropriate expression vector. The 3′ end of anucleic acid sequence encoding the first polypeptide is ligated, with orwithout a peptide linker, to the 5′ end of a nucleic acid sequenceencoding the second polypeptide ensuring the appropriate reading framesof the sequences to permit mRNA translation of the two nucleic acidsequences into a single fusion protein that retains the biologicalactivity (antigenicity) of both the first and the second polypeptides.

A peptide linker sequence may be employed to separate the first and thesecond polypeptides by a distance sufficient to ensure, that eachpolypeptide folds into its secondary and tertiary structures. Such apeptide linker sequence is incorporated into the fusion protein usingstandard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA83:8258-8262, 1986; U.S. Pat. Nos. 4,935,233 and 4,751,180. The linkersequence may be from 1 to about 50 amino acids in length. Peptidesequences are not required when the first and second polypeptides havenon-essential N-terminal amino acid regions that can be used to separatethe functional domains and prevent steric interference.

In certain embodiments of the present invention, especially for thedetection of specific antibodies directed against frameshiftpolypeptides, the frameshift polypeptides themselves may be employed.Immunogenic portions as used herein is a portion of a protein, that isrecognized by a B-cell and/or T-cell surface antigen receptor. Theimmunogenic portions comprise at least 5 amino acid residues, morepreferably at least 10 amino acid residues and most preferably at least15 amino acid residues of the frameshift polypeptides according to thepresent invention.

Immunogenic portions useful for the detection of specific antibodies maybe provided as oligopeptides or as part of larger proteins. This isdependent on the embodiment of the invention. Where antibodies are to bedetected, the antigenic epitopes may either be primary structures ofpolypeptides or the epitopes may be built by complex arrangements oftertiary structures of polypeptides. Concerning cells directed againstspecific frameshift peptides the relevant immunogenic portions aremerely short fragments of peptides with a length of about 10-20 aminoacids. Thus depending on the particular detection method the immunogenicportions have to be chosen.

In one embodiment of the invention a set of frameshift polypeptides isused for the detection of antibodies. The set may be a combination ofthe relevant peptides in solution, in cases, where information about thepresence of immunological entities in general is sought. In contrast,when information about the presence or absence of particular frameshiftpeptides within a set of peptides is sought, the frameshift peptides mayfor example be tested each as a single, simultaneously in multipletesting reactions. Such experiments may for example be carried out inform of multi-well tests or using peptide arrays etc.

In order to address a representative choice of mutations potentiallycharacterizing a disorder a set of frameshift polypeptides usedaccording to the method disclosed herein comprises for example 5-20, ina preferred embodiment 10-30, in another preferred embodiment 20-50, ina more preferred embodiment 50-100, in an even more preferred embodiment100-500 and in the most preferred embodiment more than 500 differentframeshift polypeptides originating from frameshift mutations in codingmicrosatellite regions. The frameshift polypeptides to be used as a setaccording to the present invention are selected with respect to a numberof parameters characterizing said polypeptides. A nucleic acid sequenceencoding a fusion protein of the present invention is constructed usingknown recombinant DNA techniques to assemble separate nucleic acidsequences encoding the first and second polypeptides into an appropriateexpression vector. The 3′ end of a nucleic acid sequence encoding thefirst polypeptide is ligated, with or without a peptide linker, to the5′ end of a nucleic acid sequence encoding the second polypeptideensuring the appropriate reading frames of the sequences to permit mRNAtranslation of the two nucleic acid sequences into a single fusionprotein that retains the biological activity (antigenicity) of both thefirst and the second polypeptides.

Generally using an immense or unlimited number of occurring frameshiftpeptides it would be possible to address any potential number ofdisorder, that is associated with frameshift mutations. Due to practicaland immunological concerns the number of peptides, that are included ina vaccine must be limited. To ensure a broad range of impact for anydiagnostic or therapeutic method employing a set of frameshiftpolypeptides, the selection of particular peptides has to be based onrationale considerations.

A set of frameshift polypeptides used according to the method disclosedherein comprises at least 3, 4, 5, 6 or even 7 and in certainembodiments 8, 9 or 10 frameshift polypeptides. Due to immunological aswell as practical concerns The set of frameshift peptides used as avaccine may not include an unlimited number of frameshift peptides.Preferably the set of frameshift peptides comprises at maximum 15, in amore preferred embodiment at maximum 20 and in the most preferredembodiment of the invention at maximum 30 frameshift polypeptides. Inorder to ensure a maximum range of disorders to be addressed by theselected set of frameshift polypeptides the members of the set have tobe selected by reasonable considerations. The frameshift polypeptides tobe used as a set according to the present invention are selected withrespect to a number of parameters characterizing said polypeptides.

In one embodiment the members of the set are chosen, so that only threedifferent peptides may be used to cover a wide range of tumors for aswell therapeutic as well as diagnostic approaches. Using larger panelsof frameshift peptides the stringency to the choice of individualpeptides is lowered. In especially small sets all parameters influencingthe efficacy of addressing a wide range of tumors need to be optimallymet.

In larger panels even peptides occurring with lower frequencies orallowing for efficient immune-escape may be included.

One crucial aspect influencing the selection of the frameshift peptidesis the mutation frequency of the relevant microsatellite region and thusthe frequency of a particular expressed frameshift polypeptide. Mutationfrequency as used in the context of the present invention pertains tothe percentage of samples within a defined range of total samples, whichshow a particular mutation. Any method suitable for the determination ofthe percentage of individuals in a range of samples displaying theexistence of a particular genotype or phenotype (with respect to theexpression of polypeptides) may be employed for the determination of thefrequency according to the present invention. The frequencies may bedetermined e.g. as described below in example 1.

The frequency may be the frequency of frameshift mutation within acoding microsatellite region or a frequency of expression of aframeshift polypeptide. Furthermore the frequency may be e.g. afrequency over a total of different tumor entities, the frequency withrespect to a particular tumor entity, a frequency over several tumorsentities restricted to particular stages of tumorigenesis or a frequencywith respect to a particular tumor entity restricted to particularstages of tumorigenesis.

The frequency according to the present invention has to be determinedtaking into account a range of samples sufficient to render significantdata. For example preferably at least 50 to 100 tumors may be includedin the range of samples analyzed. In case, that a smaller number ofsamples has been taken into account for determination of the frequency,a variation of the determined frequency may take place, if the range ofsamples is broadened.

Generally any frequency as determined may be used as a tool for thechoice of a set of frameshift polypeptides according to the presentinvention. To ensure best results for the method according to thepresent invention a largest possible number of samples has to be takeninto account. Yet if there are only rare data, a frequency may also bedetermined with respect to a restricted number of samples. This mayespecially be true, if the data for the restricted population of samplesindicates a quite high frequency of a particular peptide, and thusimplies a high therapeutic value for the respective peptide.

Especially in cases, where a frequency is to be determined related to aparticular tumor entity or related to particular stages of thetumorigenesis, the population of samples may be restricted.

The mutation may occur at any time during the tumor evolution and may bepersistent or may be eliminated from the genome. Thus the frequency maycomprise the frequency of the expression of a peptide at a particularstage of tumorigenesis or the occurrence of a genetic mutation at aparticular defined stage of tumorigenesis. In one embodiment of theinvention the frequency for the mutation is determined taking intoaccount the widest possible range of samples. In this embodiment themethod disclosed herein may be especially useful for the preventivevaccination of tumors. In another embodiment of the present inventionthe frequencies of mutation are related to specified tumor entities. Inthis embodiment the method according to the present invention may e.g.especially be useful for immuno-therapeutic approaches in treatment oftumors or in the preventive vaccination of particular subpopultions withan elevated risk for the occurrence of particular tumors. In a thirdembodiment of the present invention the frequency may be related toparticular stages in tumorigenesis of particular tumors. This embodimentmay be e.g. especially useful for the treatment of diagnosticallydefined tumors or for adjuvant treatment of tumors simultaneously orfollowing primary treatment of tumors.

Using frameshift polypeptides, that occur with a high frequency, theprobability, that a particular tumor expresses the peptide and may thusbe recognized by antibodies or antigen-recognizing cells, increases. Bycombination of multiple such polypeptides, the probability to addressparticular tumor cells further is raised. Preferred frameshiftpolypeptides according to the present invention occur in at least 25%,more preferred frameshift polypeptides in at least 30% and mostpreferred frameshift polypeptides in at least 33% of the cases of theparticular condition to be treated by the method according to thepresent invention.

According to the present invention the polypeptides are chosen to beexpressed with a high frequency, thus the set of polypeptides may belimited to a number of members and nonetheless may cover a range ofoccurring diseases as wide as possible. For example a set of 10polypeptides, each occurring with a frequency of more than 30 percentwill statistically cover a range of more than 95% of the potentiallyexisting disorders, as far as they have been included in the studiesleading to the respective frequencies. Using 7 polypeptides requires theapplication of polypeptides with higher frequencies in order to cover arange of about 95% of potentially existing tumors. Using a set ofpolypeptides derived from enormously frequent mutated microsatelliteregions may also allow for the employment of a set of only fivedifferent polypeptides without lowering the range of potentiallyaddressed tumors. Thus the set of polypeptides comprises in a preferredembodiment at least 5, in a more preferred embodiment at least 7 and inthe most preferred embodiment at least 10 different frameshiftpolypeptides.

A second aspect influencing the choice of a suitable set of frameshiftpolypeptides concerns the type of mutation found in the microsatelliteregion. Frameshift mutations microsatellites are usually due to DNApolymerase slippage and may be characterized by the type of the repeat.Thus in mono nucleotides repeats this type of mutation renders 1 ntinsertion or deletion. In dinucleotide repeats and tetranucleotiderepeats mutations are insertions or deletions of 2 or 4 nt respectively.(Polypeptides encoded by genes with coding microsatellites and therespective polypeptides encoded by genes with frameshift mutations aregiven in FIGS. 2-1 to 2-8) For example commonly (−1) mutations occur inmononucleotide repeats (MNRs). In these mutations one nucleotide isdeleted such that the reading frame is shifted by one nucleotide towardthe 5′ end of the gene compared to the original reading frame. This typeof mutation renders a reading frame identical to that produced by (+2)mutations, which arise from two nucleotide insertions in the respectivemicrosatellites. The respective (−1) or (+2) polypeptides might differby one amino acid. The other frameshift mutation variant leading toframeshift mutations differing from (−1) mutations concerning theresulting reading frame is the (+1) mutation, arising from onenucleotide insertion, thus rendering a reading frame shifted onenucleotide toward the 3′ end of the gene compared to the originalreading frame. This mutation type gives a reading frame identical tothat of (−2) mutations, wherein the encoded polypeptides differ by oneamino acid. (−3) and (+3) mutations are irrelevant according to thepresent invention, for they do not give rise to frameshift polypeptides.According to the present invention the polypeptides included within theset shall be selected as to cover the widest possible range of tumors.Thus a set comprises for example (−1) frameshift polypeptides andadditionally (+1) frameshift polypeptides. Using more than one possiblenovel reading frame of the particular gene broadens the spectrum oftarget cells and thus may prevent escape of particular cells from beingeliminated according to the present invention.

A further aspect influencing the choice of the member polypeptidesincluded in a set of frameshift polypeptides according to the presentinvention is the involvement of the gene encoded for by the codingsequence containing the respective microsatellite in particularbiochemical pathways. The term biochemical pathway is used with a ratherbroad meaning herein. Biochemical pathways as used within this documentshall for example include signal transduction pathways, enzymaticpathways, metabolic pathways, the apoptosis pathway, DNA repair orpolymerization pathways, the pathway of meiosis etc. To broaden thespectrum of tumors to be addressed by the set of frameshiftpolypeptides, members of different pathways are included in the set. Ina preferred embodiment at least 5 different pathways, in a morepreferred embodiment at least 4 different pathways and in the mostpreferred embodiment at least 3 different pathways are represented bythe frameshift polypeptides in a set according to the present invention.For example the TGWU as a member of a signal transduction pathway may beused in combination with the BAX gene as a member of the apoptosispathway with additional other polypeptides associated with otherpathways.

A final aspect influencing the choice of suitable frameshiftpolypeptides to be included in the set according to the presentinvention is the length of the novel (poly)peptide sequence arising fromthe mutation. The shift of the reading frame leads to novel stop codons.Thus the new peptides are not of the same length as the polypeptidesnaturally encoded by the particular gene. In most cases the new peptidesare shorter or even significantly shorter than the original wild-typepolypeptide. Frequently rather oligopeptides arise from the frameshiftmutations. The fidelity of the immune system in recognizing “foreign”molecules reaches thus far, to identify even polypeptides, that differfrom “own” polypeptides in only one single amino-acid mutation. Thefragments, bound by the antigen presenting HLA molecules comprise about12 amino acid residues. To enhance the fidelity of recognition of newpolypeptides more than one single amino-acid difference should bepresent. A polypeptide comprising 3 consecutive amino acids differingfrom the wild-type amino acid sequence is reliably recognized as foreignby the immune system. This may be due to the increased probability ofnew amino acid combinations being present in different fragmentsproduced by the antigen presenting machinery. Thus the frameshiftpolypeptides according to the present invention contain at least one newamino acid, not present in the wild-type polypeptide, in a morepreferred embodiment at least 2 new amino acids and in the mostpreferred embodiment at least 3 new amino acids.

According to the named parameters a basic set of frameshift polypeptidesmay be tailored, that is suitable to address a large variety of tumorsand minimizes the danger of escape of single tumor cells from thetherapy. Thus the probability of survival of tumor cells in an organismfollowing immuno-therapy can be minimized and the rate of recurrence ofthe cancer can be reduced.

A basic set of frameshift polypeptides includes frameshift polypeptides,that do occur with a high mutation frequency in associated disorders.Additionally the polypeptides within the set are chosen to be involvedin different biochemical pathways. The mutation types are chosen, thatpolypeptides of a minimal length of 3 amino acid residues is expressedfrom the mutated nucleic acid sequence. Furthermore different mutationtypes of one single microsatellite may be included in the set ifapplicable.

Examples of basic sets of frameshift polypeptides for use in therapeuticas well as diagnostic methods of the present invention include:

Set1: HT001 U79260 MACS Set2: HT001 TAF1B MACS Set3: HT001 TGFB2R MACSSet4: HT001 U79260 TGFB2R Set5: HT001 U79260 TAF1B Set6: HT001 TGFB2RTAF1B Set7: HT001 U79260 TGFB2R MACS Set8: HT001 U79260 TGFB2R AC1 Set9:HT001 U79260 TGFB2R TAF1B Set10: HT001 TGFB2R MACS CASP5 Set11: HT001U79260 MACS CASP5 Set12: HT001 U79260 MACS AC1 Set13: HT001 TGFB2R TAF1BCASP5 Set14: HT001 U79260 MACS OGT Set15: U79260 TGFB2R AC1 CASP5 Set16:HT001 U79260 TGFB2R MACS AC1 Set17: HT001 U79260 TGFB2R TAF1B MACSSet18: HT001 U79260 TGFB2R TAF1B AC1 Set19: HT001 U79260 TGFB2R MACSAIM2 Set20: HT001 U79260 TGFB2R TAF1B AIM2 Set21: U79260 TGFB2R TAF1BAC1 CASP5 Set1: HT001 U79260 TGFB2R AC1 CASP5 Set22: U79260 TGFB2R MACSAC1 CASP5 Set23: HT001 U79260 TAF1B MACS AC1 Set24: HT001 U79260 TAF1BMACS CASP5 Set1: HT001 U79260 MACS AC1 OGT Set25: HT001 U79260 MACS MSH3OGT Set26: HT001 U79260 TGFB2R MACS OGT Set27: HT001 TGFB2R TAF1B AC1CASP5 Set28: HT001 U79260 TGFB2R AC1 AIM2

Additional to the parameters given above, data concerning the particulardisorder in focus are taken into account for the design of particularsets of frameshift peptides according to the present invention. Thusindividual mutation frequencies, typical mutation types, relevantbiochemical pathways or special immunological characteristics maycontribute to the set to be used in particular cases. Furthermore inparticular cases based on results of examination of particular samplesof individuals vaccines may be tailored as to optimally fit the therapyof the respective disorder. In one embodiment of the present inventionindividual tumor vaccine compositions may be set up according tomolecular profiling of individual tumors

Choosing suitable combinations of the peptides within the mixture offrameshift peptides thus enables for generation of therapeutic ordiagnostic preparations with a wide range of applicability. E.g.vaccines that elicit immune response specifically for tumors ofparticular organs. On the other hand it is possible to design sets offrameshift polypeptides that cover a wide range of degenerativedisorders or cancers in individuals. The first possibility may e.g. beespecially useful for the design of curative treatment, whereas thesecond variant of sets may be of special interest for the design ofpreventive vaccines.

The compositions and methods according to the present invention may beapplied to any eukaryotic organisms exhibiting an immunologic defencesystem. The eukaryotic organisms are for example animals of agriculturalvalue such as pigs, cows, sheep, etc., companion animals, such as cats,dogs, horses etc., animals employed in research purposes such as mice,rats, rabbits, hamsters etc. or humans.

Therapeutic methods for use in the present invention compriseimmunogenic treatment such as vaccination therapy or generallyimmuno-therapy. The vaccines for use in the present invention comprisefor example one or more sets of frameshift polypeptides.

According to the present invention frameshift polypeptides that comprisean immunogenic portion may be used for immuno-therapy for the treatmentof cancer. Immunotherapy may be broadly classified into either active orpassive immunotherapy. In active immunotherapy, treatment relies on thein vivo stimulation of the endogenous host immune system to reactagainst tumors with the administration of immune response-modifyingagents (for example, tumor vaccines, bacterial adjuvants, and/orcytokines). A patient may be afflicted with disease, or may be free ofdetectable disease. Accordingly, the compounds disclosed herein may beused to treat cancer or to inhibit the development of cancer. Thecompounds are preferably administered either prior to or followingprimary treatment of tumors such as surgical removal of the tumors,treatment by administration of radiotherapy and/or conventionalchemotherapeutic drugs or any other mode of treatment of the respectivecancer or its precursors.

In passive immunotherapy, treatment involves the delivery of biologicreagents with established tumor-immune reactivity (such as effectorcells or antibodies) that can directly or indirectly mediate antitumoreffects and does not necessarily depend on an intact host immune system.Examples of effector cells include T lymphocytes (for example, CD8+cytotoxic T-lymphocytes, CD4+ T-helper, tumor-infiltrating lymphocytes),killer cells (such as Natural Killer cells, lymphokine-activated killercells), B cells, or antigen presenting cells (such as dendritic cellsand macrophages) expressing the disclosed antigens. The polypeptidesdisclosed herein may also be used to generate antibodies oranti-idiotypic antibodies (as in U.S. Pat. No. 4,918,164), for passiveimmunotherapy.

The predominant method of procuring adequate numbers of T-cells foradoptive transfer immunotherapy is to grow immune T-cells in vitro.Culture conditions for expanding single antigen-specific T-cells toseveral billion in number with retention of antigen recognition in vivoare well known in the art. These in vitro culture conditions typicallyutilize intermittent stimulation with antigen, often in the presence ofcytokines, such as IL-2, and non-dividing feeder cells. As noted above,the immunoreactive polypeptides described herein may be used to rapidlyexpand antigen-specific T-cell cultures in order to generate sufficientnumber of cells for immunotherapy. In particular, antigen-presentingcells, such as dendritic-, macrophage- or B-cells, may be pulsed withimmunoreactive polypeptides or transfected with a nucleic acidsequence(s), using standard techniques well known in the art. Forexample, antigen presenting cells may be transfected with a nucleic acidsequence, wherein said sequence contains a promoter region appropriatefor increasing expression, and can be expressed as part of a recombinantvirus or other expression system. For cultured T-cells to be effectivein therapy, the cultured T-cells must be able to grow and distributewidely and to survive long term in vivo. Studies have demonstrated thatcultured T-cells can be induced to grow in vivo and to survive long termin substantial numbers by repeated stimulation with antigen supplementedwith IL-2 (see, for example, Cheever, M., et al, “Therapy With CulturedT-Cells: Principles Revisited,” Immunological Reviews, 157:177, 1997).

According to the present invention sets of frameshift polypeptides maybe employed to generate and/or isolate tumor-reactive T-cells, which canthen be administered to the patient. In one technique, antigen-specificT-cell lines may be generated by in vivo immunization with shortpeptides corresponding to immunogenic portions of the disclosedpolypeptides. The resulting antigen specific CD8+ CTL or CD4+T-helpercells clones may be isolated from the patient, expanded using standardtissue culture techniques, and returned to the patient.

Alternatively, peptides corresponding to immunogenic portions of thepolypeptides of the invention may be employed to generate tumor reactiveT-cell subsets by selective in vitro stimulation and expansion ofautologous T-cells to provide antigen-specific T-cells which may besubsequently transferred to the patient as described, for example, byChang et al. (Crit. Rev. Oncol. Hematol., 22(3), 213, 1996). Cells ofthe immune system, such as T-cells, may be isolated from the peripheralblood of a patient, using a commercially available cell separationsystem, such as CellPro Incorporated's (Bothell, Wash.) CEPRATE™ system(see U.S. Pat. Nos. 5,240,856; 5,215,926; WO 89/06280; WO 91/16116 andWO 92/07243). The separated cells are stimulated with one or more of theimmunoreactive polypeptides contained within a delivery vehicle, such asa microsphere, to provide antigen-specific T-cells. The population oftumor antigen-specific T-cells is then expanded using standardtechniques and the cells are administered back to the patient.

In another embodiment, T-cell receptors and/or antibodies specific forthe polypeptides can be cloned, expanded, and transferred into othervectors or effector cells for use in adoptive immunotherapy.

In a further embodiment, syngeneic or autologous dendritic cells may bepulsed with peptides corresponding to at least an immunogenic portion ofa polypeptide disclosed herein. The resulting antigen-expressing and/orpresenting dendritic cells may either be transferred into a patient, oremployed to stimulate T-cells to provide antigen-specific T-cells whichmay, in turn, be administered to a patient. The use of peptide-pulseddendritic cells to generate antigen-specific T-cells and the subsequentuse of such antigen-specific T-cells to eradicate tumors in a murinemodel has been demonstrated by Cheever et al, (Immunological Reviews,157:177, 1997).

Monoclonal antibodies directed against frameshift peptides presented oncellular membranes may according to the present invention also be usedas therapeutic compounds in order to diminish or eliminate tumors. Theantibodies may be used on their own (for instance, to inhibitmetastases) or coupled to one or more therapeutic agents. Suitableagents in this regard include radionuclides, differentiation inducers,drugs, toxins, and derivatives thereof. Preferred radionuclides include90Y, 1231, 1251, 1311, 186Re, 188Re, 211At, and 212Bi. Preferred drugsinclude methotrexate, and pyrimidine and purine analogues. Preferreddifferentiation inducers include phorbol esters and butyric acid.Preferred toxins include ricin, abrin, diptheria toxin, cholera toxin,gelonin, Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviralprotein.

Pharmaceutical compositions useful in immuno-therapy according to thepresent invention may comprise a set of at least 3, 4, 5, 6, 7, 8, 9 or10 frameshift polypeptides (or variants thereof). Moreover sets offrameshift polypeptides may comprise 5 to 20, 7 to 30 or even ore than15, 20 or 30 frameshift peptides (or variants thereof). (or variantsthereof), In certain embodiments the pharmaceutical compositionscomprise the frameshift polypeptides and a physiologically acceptablecarrier. The vaccines may additionally comprise a non-specificimmune-response enhancer, wherein the non-specific immune responseenhancer is capable of eliciting or enhancing an immune response to anexogenous antigen. Examples of non-specific-immune response enhancersinclude adjuvants, biodegradable microspheres (e.g., polylacticgalactide) and liposomes (into which the polypeptide is incorporated).Pharmaceutical compositions and vaccines may also contain other epitopesof tumor antigens, either incorporated into a fusion protein or presentwithin a separate polypeptide.

Alternatively, a pharmaceutical composition or vaccine suitable forimmunotherapy according to the present invention may contain nucleicacids, that code for one or more frameshift polypeptides according tothe present invention. Nucleic acids may for example includesingle-stranded (sense or antisense) or double-stranded molecules, andmay be DNA (genomic, cDNA or synthetic) or RNA. RNA molecules compriseas well HnRNA (containing introns) as mRNA (not containing introns).According to the present invention the polynucleotides may also belinked to any other molecules, such as support materials or detectionmarker molecules, and may, but need not, contain additional coding ornon-coding sequences. The nucleic acid may be administered in a way thatallows the polypeptides to be generated in situ. Suitable expressionsystems are known to those skilled in the art. The expression of thepolypeptides may for example be persistent or transient. Inpharmaceutical compositions and/or vaccines, providing for in-situexpression of polypeptides, the nucleic acids may be present within anysuitable delivery system known to those of ordinary skill in the art,including nucleic acid expression systems, bacteria and viral expressionsystems.

Appropriate nucleic acid expression systems comprise the necessaryregulatory nucleic acid sequences for expression in the patient, such assuitable promoters, terminators etc. Bacterial delivery systems may forexample employ the administration of a bacterium that expresses anepitope of a cell antigen on its cell surface. In a preferredembodiment, the nucleic acid may be introduced using a viral expressionsystem such as e.g., vaccinia virus, retrovirus, or adenovirus, whichmay involve the use of a non-pathogenic, replication competent virus.Suitable systems are known to those of ordinary skill in the art and aredisclosed, for example, in Fisher-Hoch et al., PNAS 86:317-321, 1989;Flexner et al., Ann. N.Y. Acad Sci. 569:86-103, 1989; Flexner et al.,Vaccine 8:17-21, 1990; U.S. Pat. Nos. 4,603,112, 4,769,330, and5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP0,345,242; WO 91/02805; Berkner, Biotechniques 6:616-627, 1988;Rosenfeld et al., Science 252:431-434, 1991; Kolls et al., PNAS91:215-219, 1994; Kass-Eisler et al., PNAS 90:11498-11502, 1993; Guzmanet al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir. Res.73:1202-1207, 1993.

In another embodiment transgenic mammalian cells may be used fordelivery and/or expression of the nucleic acids. The methods forproducing nucleic acid constructs suitable for in-situ expression ofpolypeptides are known to those of skill in the art.

Furthermore the nucleic acid may be administered as naked nucleic acids.In this case appropriate physical delivery systems, which enhance theuptake of nucleic acid may be employed, such as coating the nucleic acidonto biodegradable beads, which are efficiently transported into thecells. Administration of naked nucleic acids may for example be usefulfor the purpose of transient expression within a host or host cell.

The pharmaceutical compositions used for immuno-therapy according to thepresent invention may be administered by any suitable way known to thoseof skill in the art. The administration may for example compriseinjection, such as e.g., intra-cutaneous, intramuscular, intravenous orsubcutaneous injection, intranasal administration for example byaspiration or oral administration. A suitable dosage to ensure thepharmaceutical benefit of the treatment should be chosen according tothe parameters, such as age, sex, body weight etc. of the patient, knownto those of skill in the art.

The type of carrier to be employed in the pharmaceutical compositions ofthis invention, will vary depending on the mode of administration. Forparenteral administration, such as subcutaneous injection, the carrierpreferably comprises water, saline, alcohol, a lipid, a wax and/or abuffer. For oral administration, any of the above carriers or a solidcarrier, such as mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, talcum, cellulose, glucose, sucrose, and/or magnesiumcarbonate, may be employed. Biodegradable microspheres (e.g., polylacticglycolide) may also be employed as carriers for the pharmaceuticalcompositions of this invention. Suitable biodegradable microspheres aredisclosed, for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109. Anyof a variety of immune-response enhancers may be employed in thevaccines of this invention. For example, an adjuvant may be included.Most adjuvants contain a substance designed to protect the antigen fromrapid catabolism, such as aluminum hydroxide or mineral oil, and anonspecific stimulator of immune response, such as lipid A, Bordetellapertussis or Mycobacterium tuberculosis. Such adjuvants are commerciallyavailable as, for example, Freund's Incomplete Adjuvant and CompleteAdjuvant (Difco Laboratories, Detroit, Mich.) and Merck Adjuvant 65(Merck and Company, Inc., Rahway, N.J.).

The pharmaceutical compositions or immuno-therapeutic methods accordingto the present invention may be used for the treatment of degenerativedisorders or cancers. For example the compositions and methods may beemployed in the therapy of diagnosed cancers in order to eliminate thetumor cells from the affected organism. As well primary tumors asmetastases or disseminated tumor cells within an organism may be targetsto the therapeutic compounds and methods disclosed herein.

Furthermore the compositions and methods of the invention may beemployed in the treatment of pre-neoplastic conditions. In this case thepre-neoplastic cells or tissues may be directly addressed by theimmuno-therapeutic compositions or methods, or may be hindered fromevolving into neoplastic or dysplastic conditions. For example in thiscase the pre-neoplastic condition may be treated preventively. By thevaccination the immune response may be elicited, so that emergingneoplasms may be destroyed.

The methods and compositions according to the present invention may alsobe used for the prevention of degenerative disorders or cancersassociated with frameshift mutations in coding microsatellites. For thispurpose a vaccination of a population of organisms or of subgroups ofsaid population may be performed. The subgroups may be built by suitableparameters such as hereditary predisposition for the emergence ofdegenerative disorders, exposure to factors, that increase the risk ofbeing affected by said disorders etc.

In one embodiment of the present invention the peptides disclosed hereinmay be used for the diagnosis of disorders associated with frameshiftmutations in coding microsatellite regions.

Diagnosis as used in the context of the present invention may comprisedetermining the presence or absence and/or the level of frameshiftpeptides or of specific immunological entities directed againstparticular frameshift peptides in a sample, and assessing diagnosis fromsaid presence or absence and/or level of frameshift peptides and/orimmunological entities specifically directed against said frameshiftpolypeptides.

Based upon the determined presence or absence and/or the levels offrameshift peptides or of immunological entities specifically directedagainst particular frameshift peptides in the samples individuals can besubdivided into subgroups. The subgroups may be created according toscientific or clinical data, such as e.g. survival, recurrence ofdisease, frequency of metastases etc., related to the presence orabsence and/or levels of frameshift peptides or of particular frameshiftpeptides in samples of tissues affected with a particular disorder, oftissues being in question of being affected with a particular disorderor of tissues at risk of being affected with a particular disorder.

Based upon these subgroups an assessment of prognosis may be done.According to the subgroups the therapy of the individuals affected bythe disorders (e.g. tumors) may be tailored.

Monitoring may comprise detecting the presence or absence or level offrameshift peptides or of immunologic entities specifically directedagainst frameshift polypeptides in samples taken at different points intime and determining the changes in said levels or presences orabsences. According to said changes the course of the disease can befollowed. E.g. the occurrence of frameshift peptides or of immunologicentities directed against frameshift peptides, that have not beenpresent at an earlier time-point may be indicative of the progression ofevolution of the affected tissue. The course of the disease may be usedto select therapy strategies for the particular individual.

Another aspect of diagnosis and monitoring of the disease courseaccording to the present invention may comprise the detection of minimalresidual disease. This may comprise for example the detection ofpresence and/or level of frameshift peptides or of immunologic entitiesspecifically directed against said frameshift polypeptides, that havenot been present in earlier examinations in one or more body samplesfollowing initial therapy of an individual once or at several timepoints. According to the presence and/or level of frameshift peptides orof immunologic entities specifically directed against new frameshiftpolypeptides detected in the samples one may select a suitable therapyfor the particular individual.

Furthermore the diagnostic method may be carried out to detectdisseminated tumor cells in biological samples as MRD diagnosis ofminimal residual disease. For this purpose the detection of the level ofimmunological entities or the presence of frameshift peptides or ofimmunological entities, specific for particular frameshift peptides,that have not been detected in prior examinations, may be performed.

Immunological entities as used in the context of the present inventionshall comprise any components of the mammalian immune system, that areable to specifically react with an antigenic epitope. Such immunologicalentities may comprise for example antibodies, all immunoglobulins, suchas e.g. IgG, IgM, IgA, IgE, IgD, specific CD8+ T-cells or specificT-helper cells.

A sample according to the method of the present invention may compriseany sample comprising frameshift peptides or immunological entities asdefined above. Samples may comprise samples of clinical relevance, suchas e.g. secretions, smears, body fluids, urine, semen, stool, bile,biopsies, cell- and tissue-samples. Biopsies as used in the context ofthe present invention may comprise e.g. resection samples of tumors,tissue samples prepared by endoscopic means or needle biopsies oforgans.

Such samples may comprise for example intact cells, lysed cells or anyliquids containing polypeptides, antibodies, immunoglobulins or cellsspecifically directed against frameshift peptides. Even solids, to whichpeptides, cells, cell fragments or antigen binding polypeptides, such asantibodies or immunoglobulins may adhere, or may be fixed to, may besamples according to the method disclosed herein. The method fordetection of the level of the frameshift peptides or of theimmunological entities according to the present invention is any method,which is suited to detect very small amounts of specific frameshiftpeptides or of specific immunological entities in biological samples.The detection reaction according to the present invention is a detectioneither on the level of polypeptides, nucleic acids, antibodies or on thelevel of cells specific for particular antigens.

For diagnostic purposes detection procedures related to one singleframeshift polypeptides or to immunological entities specificallyrecognizing said frameshift peptides may be performed. Furthermoredetection procedures may be performed that are tailored to display thepresence or absence or the level of one or more sets of polypeptides orimmunological entities directed against these polypeptides, wherein thesets have been put together according to rational combinatorialparameters as they are given below.

The detection may be carried out in solution or using reagents fixed toa solid phase. Solid phases may comprise beads of a variety ofmaterials, such as e.g. agarose, dextrane polymers, polystyrene, silica,etc. or surfaces of suitable materials such as e.g. polystyrene, glass,agarose, protein, dextran etc. coated surfaces etc. The detection of oneor more immunological entities, such as immunoglobulins or cellscarrying specific antigen recognizing epitopes, with different antigenbinding specificities may be performed in a single reaction mixture orin two or more separate reaction mixtures. Alternatively the detectionreactions for several immunological entities may for example beperformed simultaneously in multi-well reaction vessels.

Applicable formats for the detection reaction according to the presentinvention may be, (reverse) blotting techniques, such as Western-Blot.The blotting techniques are known to those of ordinary skill in the artand may be performed for example as electro-blots, semidry-blots,vacuum-blots or dot-blots. Furthermore immunological methods fordetection of molecules may be applied, such as for exampleimmunoprecipitation or immunological assays, such as ELISA, RIA, Elispotassay, lateral flow assays, immuno-cytochemical methods etc.

The immunological entities specifically recognizing particularframeshift peptides may be detected using reagents that specificallyrecognise these immunological entities alone or in complex with theirrespective antigen (e.g. antibodies), or reagents, that are specificallyrecognized by the immunological entities themselves (e.g. the antigen).In one embodiment the antigen may be fused to another polypeptide, so asto allow binding of the antigen by the immunological entity in questionand simultaneously binding of the second part of the fusion protein byanother (labelled) antibody for the detection. The detection reactionfor the immunological entities may comprise one or more reactions withdetecting agents either recognizing the initial entities or recognizingthe prior molecules used to recognize the immunological entities.

The detection reaction further may comprise a reporter reactionindicating the presence or absence and/or the level of the immunologicalentities. The reporter reaction may be for example a reaction producinga coloured compound, a bioluminescence reaction, a chemiluminescentreaction, a fluorescence reaction, generally a radiation emittingreaction etc. The detection reactions may for example employ antibodiesor binding reagents that are detectably labelled.

Furthermore the binding of detection molecules to the entities inquestion may be detected by any measurable changes in physical orphysical-chemical properties, such as changes in spectroscopicproperties, in magnetic resonance properties etc. Differentimmunological entities or immunological entities of differentspecificities may be recognized by different methods or agents. This maybe due to difficulties in detection of several entities, or entitieswith particular specificities by a particular method. An advantage ofthe use of different detection techniques for different immunologicalentities or for immunological entities with different specificities mayfor example be, that the different reporter signals referring todifferent immunological entities could be distinguished.

Generally in a method according to the present invention the detectionof different immunological entities such as the detection ofimmunoglobulins and the detection of immunocompetent cells may beperformed simultaneously.

For all detection purposes optionally the original sample may beconcentrated by any suitable means known to those of ordinary skill inthe art. Furthermore steps may be involved to selectively extractimmunological entities from the sample mixture such as affinity basedpurification techniques either employing specific antibodies or therespective antigen recognized by the entities in question.

In one preferred embodiment of the invention the detection of the levelof immunological entities specific for frameshift peptides is carriedout on the level of antibodies. This may be e.g. performed using thespecific interaction between the respective frameshift peptides with theantibodies. The determination of the presence or absence and/or thelevel of the antibodies may for example be carried out withrecombinantly produces frameshift peptides. The peptides can be used inmany different detection techniques for example in western-blot, ELISAor immuno-precipitation. In one embodiment the detection of antibodiesis carryout as antibody capture assay (Antibodies A laboratory Manual,Harlow, Ed. et al., Cold Spring Harbor Laboratory 1988).

In another embodiment of the invention the detection of the specificantibodies is carried out using monoclonal or polyclonal antibodiesspecifically recognizing the antigen binding epitope of the firstantibodies. For this purpose the above mentioned immunological detectionprocedures may be applied. In a further embodiment chimeric antigens maybe employed in the detection reaction. Such chimeric antigens may forexample comprise fusion proteins combining the antigenic epitope of aframeshift polypeptide, recognized by the antibody in question, fused toanother antigen, that may be recognized by a detection antibody. Theparticular antigens within the chimeric polypeptide may be separated bya linker or spacer region.

Any other method for determining the amount of particular antibodies orimmunoglobulins in biological samples can be used according to thepresent invention.

Generally the detection of the antibodies according to the presentinvention may be performed as well in vitro as directly in situ forexample in the course of an immuno-histochemical or immuno-cytochemicalstaining reaction.

Cells exhibiting specificity for a particular antigen may be detected byany methods suitable for that purpose known to those of ordinary skillin the art. Methods may for example comprise proliferation-assays,cytokine-ELISAs, ELISpot assays, intracellular FACS-staining,PCR-mediated identification of peptide-specific cytokine (orsimilar)—expressing cells, tetramer-staining, cytotoxicity assays andDTH-(delayed type hypersensitivity) reactions.

In case of proliferation-assays induction of peptide-specific T-cellproliferation may be measured by methods known to those of skill in theart. This can be achieved by simply counting of cells, by measuringincorporation of labelled nucleotides into cellular DNA or by measuringlevel and/or activity of cellular protein(s). Cytokine-ELISA maycomprise identification of peptide-specific cytokine-secreting cells bymeasuring cytokine levels in supernatant. In the course of an ELISpotassay the number of peptide-specific cytokine (i.e. IFN-g)-secretingcells in a sample is determined. Similarly the IntracellularFACS-staining identifies cytokine-expressing cells on the protein level.In contrast (real-time) PCR may be used for identification ofpeptide-specific cytokine (or similar)—expressing cells on thetranscript level. In the course of a tetramer-staining assay the labelis a tetramer-molecule of recombinant MHC-class I molecules, loaded withspecific peptide and coupled to a dye. The tetramer binds to the T-cellreceptor. Cytotoxicity assays are a method for identification of cells,that can recognize and kill target cells in a peptide-specific manner.DTH-(delayed type hypersensitivity) reaction is based on the measuringof skin reaction of vaccinated persons after intradermal (or similar)application of peptide(s).

In a preferred embodiment of the invention the detection of theimmunological entities directed against particular frameshiftpolypeptides is carried out on the level of antibodies. In thisembodiment the binding agent may be for example a frameshift polypeptideor a fragment thereof, recognized by the respective antibody, or afusion polypeptide comprising said frameshift polypeptide or a fragmentthereof. Furthermore the binding agent may comprise an antibody or afragment thereof specific for the antibody in question, for the complexof the antibody with the respective frameshift polypeptide or for anantigenic epitope fused to the frameshift polypeptide.

In another embodiment of the test kit the detection of the immunologicalentities is carried out on the level of cells specifically recognizingframeshift polypeptides. In this embodiment of the invention the reagentfor the detection may be for example a frameshift polypeptide or afragment thereof, recognized by the respective antibody or T-cellreceptor, or a fusion polypeptide comprising said frameshift polypeptideor a fragment thereof. Furthermore the binding agent may comprise anantibody or a fragment thereof specific for the antibody in question,for the complex of the antibody with the respective frameshiftpolypeptide or for an antigenic epitope fused to the frameshiftpolypeptide.

The method for detection of the level of the frameshift polypeptidesaccording to the present invention is any method, which is suited todetect very small amounts of specific biologically active molecules inbiological samples. The detection reaction according to the presentinvention is a detection either on the level of nucleic acids or on thelevel of polypeptides.

The detection may be carried out in solution or using reagents fixed toa solid phase. The detection of one or more molecular markers, such aspolypeptides or nucleic acids, may be performed in a single reactionmixture or in two or separate reaction mixtures. Alternatively thedetection reactions for several marker molecules may for example beperformed simultaneously in multi-well reaction vessels. The markerscharacteristic for the frameshift polypeptides may be detected usingreagents that specifically recognise these molecules. The detectionreaction for the marker molecules may comprise one or more reactionswith detecting agents either recognizing the initial marker molecules orrecognizing the prior molecules used to recognize other molecules.

In one preferred embodiment of the invention the detection of the levelof frameshift polypeptides is carried out by detection of the level ofnucleic acids coding for the frameshift polypeptides or fragmentsthereof present in the sample. The means for detection of nucleic acidmolecules are known to those skilled in the art. The procedure for thedetection of nucleic acids can for example be carried out by a bindingreaction of the molecule to be detected to complementary nucleic acidprobes, proteins with binding specificity for the nucleic acids or anyother entities specifically recognizing and binding to said nucleicacids. This method can be performed as well in vitro as directly in situfor example in the course of a detecting staining reaction. The use ofthis detection procedure is restricted to cases, where hybridisationproperties of the respective frameshift mutations are significantlyaltered in comparison to the respective wild-type nucleic acids. Anotherway of detecting the frameshift polypeptides in a sample on the level ofnucleic acids performed in the method according to the present inventionmay comprise an amplification reaction of nucleic acids. In these casesa subsequent reaction displaying the presence or absence of a frameshiftmutation within the coding microsatellite region is necessary.

In another preferred embodiment of the invention the detection of thelevel of frameshift polypeptides is carried out by determining the levelof expression of a protein. The determination of the frameshiftpolypeptides on the protein level can for example be carried out in areaction comprising an antibody specific for the detection of theframeshift polypeptides. The antibodies can be used in many differentdetection techniques for example in western-blot, ELISA orimmuno-precipitation. Generally antibody based detection can be carriedout as well in vitro as directly in situ for example in the course of animmuno-histochemical staining reaction. Any other method for determiningthe amount of particular polypeptides in biological samples can be usedaccording to the present invention.

Furthermore according to the present invention diagnosis may comprisedetection of immunological entities specifically recognizing particularframeshift peptides using reagents that specifically recognise theseimmunological entities alone or in complex with their respective antigen(e.g. antibodies), or reagents, that are specifically recognized by theimmunological entities themselves (e.g. the antigen). In one embodimentthe antigen may be fused to another polypeptide, so as to allow bindingof the antigen by the immunological entity in question andsimultaneously binding of the second part of the fusion protein byanother (labelled) antibody for the detection. The detection reactionfor the immunological entities may comprise one or more reactions withdetecting agents either recognizing the initial entities or recognizingthe prior molecules used to recognize the immunological entities.

The detection reaction further may comprise a reporter reactionindicating the presence or absence and/or the level of the frameshiftpolypeptides or of the immunological entities. The reporter reaction maybe for example a reaction producing a coloured compound, abioluminescence reaction, a chemiluminescent reaction, a fluorescencereaction, generally a radiation emitting reaction etc. The detectionreactions may for example employ antibodies or binding reagents that aredetectably labelled.

Furthermore the binding of detection molecules to the peptides orimmunological entities in question may be detected by any measurablechanges in physical or physical-chemical properties, such as changes inspectroscopic properties, in magnetic resonance properties etc.Different polypeptides or immunological entities or immunologicalentities of different specificities may be recognized by differentmethods or agents. This may be due to difficulties in detection ofseveral entities, or entities with particular specificities by aparticular method. An advantage of the use of different detectiontechniques for different polypeptides and/or immunological entities orfor immunological entities with different specificities may for examplebe, that the different reporter signals referring to differentimmunological entities could be distinguished.

Generally in a method according to the present invention the detectionof different polypeptides, of different immunological entities such asthe detection of immunoglobulins and the detection of immunocompetentcells may be performed simultaneously.

For all detection purposes optionally the original sample may beconcentrated by any suitable means known to those of ordinary skill inthe art. Furthermore steps may be involved to selectively extractpolypeptides and/or immunological entities from the sample mixture suchas affinity based purification techniques either employing specificantibodies or the respective antigen recognized by the entities inquestion.

Another aspect of the present invention is a testing kit for performingthe method according to the present invention. The kit may be forexample a diagnostic kit or a research kit.

A kit according to the present invention comprises at least an agentsuitable for detecting the immunological entities according to themethod disclosed herein. Furthermore a kit according to presentinvention may comprise:

a) reagents for the detection of the antibodies or cells specificallyrecognizing antigens.

b) reagents and buffers commonly used for carrying out the detectionreactions as described herein, such as buffers, detection-markers,carrier substances and others

c) a sample for carrying out a positive control reaction, that maycomprise an antigen or a set of antigens, to which all members of atarget population of individuals have antibodies.

The reagent for the detection of the antibodies or cells specificallyrecognizing antigens may include any agent capable of binding to theantibodies or cells specifically recognizing antigens. Such reagents mayinclude proteins, polypeptides, nucleic acids, peptide nucleic acids,glycoproteins, proteoglycans, polysaccharides or lipids.

The sample for carrying out a positive control may comprise for examplean antigenic peptide or a set of antigenic peptides. Suitable antigensmay include antigens, against which a wide percentage of the populationhas antibodies. Examples of such antigens may for example compriseantigens present in lysed E. coli cells, tetanus antigen, the viralcapsid antigen of the Epstein-Barr virus, antigens derived from matrixproteins of Haemophilus influenzae. The antigens may for example be usedas a mixture to ensure, that a particular individual actually displays apositive reaction.

The present invention provides compositions and methods for enhancedimmunotherapy of disorders associated with MSI⁺ related occurrence offrameshift peptides. The invention provides sets for the immuno-therapyof said disorders, that address a wide range of different types ofdisorders in an organism and may thus be employed as a preventivevaccine against said disorders. Furthermore the invention also providessets of polypeptides useful for the treatment of particular types ofdisorders. The use of sets of frameshift polypeptides for theimmuno-therapy according to the present invention provides the means fora reliable treatment of degenerative disorders and cancers associatedwith frameshift mutations in coding microsatellites reducing the risk ofescape of several tumor cells or of a population of tumor cells frombeing addressed by the therapy. Thus the method of the invention reducesthe risk of recurrence of tumors after immuno-therapy employing CTLs,T-helper cells and possibly specific antibody producing B-cells raisedagainst frameshift polypeptides characteristic for tumor cells. Thepresent invention furthermore provides compositions and methods forenhanced diagnosis and therapy of disorders associated with MSI⁺ relatedoccurrence of frameshift peptides. The invention provides new frameshiftpolypeptides for diagnosis therapy of said disorders, that may be usedeach alone or in combinations tailored, to address a wide range ofdifferent types of disorders in an organism and may thus be employed asa preventive vaccine against said disorders.

The following examples are given for illustration of the invention onlyand are not intended to limit the scope of the invention.

EXAMPLES Example 1: Analysis of the Mutation Frequency of GenesHarbouring Repeat Tracts

Investigations were performed regarding the mutation frequencies ofunpublished coding microsatellite regions. Nine novel codingmicrosatellites residing in genes not yet analyzed for frameshiftmutations in MSI colorectal, endometrial or gastric tumors have beenexamined in the course of the studies leading to the present invention.They include three genes containing A11 repeats (TAF1B, MACS, HT001),five genes with A10 repeats (CHD2, UVRAG, TCF6L1, ABCF1, AIM2) and onegene harboring a G9 repeat (ELAVL3). The MSI status of these specimenwas determined using the NCI ICG-HNPCC microsatellite reference markerpanel (8). PCR reactions were performed as follows:

Genomic DNA was isolated from 5-8 haematoxylin and eosin stained 5 μmsections after microdissection, using the Qiamp Tissue Kit (Qiagen,Hilden, Germany). Preparation of DNA from cell lines was performedaccording to standard protocols. PCR primers were designed to closelyflank the target sequence, yielding short amplimeres of about 100 basepairs thus allowing precise fragment sizing and robust amplificationfrom archival tissues (Table 1). PCR reactions were performed in a totalvolume of 25 μl containing 50 ng genomic DNA, 2.5 μl 10× reaction buffer(Life Technologies, Karlsruhe, Germany), 1.5 mM MgCl₂, 200 μM dNTPs, 0.3μM of each primer, and 0.5 U Taq DNA polymerase (Life Technologies) andusing the following conditions: initial denaturation at 94° C. for 4min, followed by 35 cycles of denaturation at 94° C. for 30 s, annealingat 58° C. for 45 s, and primer extension at 72° C. for 30 s. The finalextension step was carried out at 72° C. for 6 min. PCR fragments wereanalyzed on an ALF DNA sequencing device (Amersham Pharmacia Biotech,Freiburg, Germany) using 6.6% polyacrylamide/7 M urea gels. Size, heightand profile of microsatellite peaks were analyzed using the AlleleLinkssoftware (Amersham Pharmacia Biotech). Coding microsatellite instabilitywas scored, if smaller or larger-sized amplimeres were detected in tumorDNA compared to DNA from non-neoplastic cells. Allele intensities weredetermined and ratios of wild-type and novel alleles in normal and tumortissues were calculated, defining a two-fold difference as threshold forallelic shifts. Similarly, unstable alleles in tumor cell lines wereidentified by comparison to 36 unmatched normal mucosae. In order todetermine the predicted repeat type and length amplified codingmicrosatellites were subjected to Big Dye terminator cycle sequencing(Perkin Elmer, Darmstadt, Germany) and subsequent analysis on an ABI 310sequencing device.

The frequencies of mutations in a particular microsatellite regionexamined herein are given in Tables 1 and 2. The mutation rates arecalculated with respect to the total number of samples included in thestudy.

TABLE 1 Coding microsatellite genes investigated in MSI-H colorectalcancer. Repeat: type of nucleotide and length of repeat tract, n: numberof samples investigated, mut.: number of samples mutated, %: percentageof mutated samples, gene acc.no. repeat n mut. % HPDMPK Y10936 T14 21 2095% HT001 AF113539 A11 20 17 85% TGFbIIR D50683 A10 735 602 82% U79260U79260 T14 21 17 81% PTHL3 M24350 A11 21 17 81% MACS D10522 A11 19 1474% TAF1B L39061 A11 19 13 68% AC1 D82070 T10 21 14 67% AIM2 AF024714A10 19 10 53% BAX L22473 G8 538 235 44% SLC23A1 AF058319 C9 21 9 43%ABCF1 AF027302 A10 19 8 42% TCF-4 Y11306 A9 248 98 40% Caspase 5 U28015A10 120 47 39% TCF6L1 M85079 A10 18 7 39% FLT3LG U29874 C9 21 8 38% MSH3J04810 A8 596 223 37% ELAVL3 D26158 G9 19 7 37% MAC30X L19183 A10 21 733% UVRAG X99050 A10 18 6 33% SLC4A3 U05596 C9 21 7 33% GRB-14 L76687 A957 18 32% RIZ U17838 A9 83 23 28% MBD4/MED1 AF072250 A10 83 22 27% RAD50U63139 A9 109 28 26% MSH6 U54777 C8 684 169 25% IGFIIR Y00285 G8 423 9021% Axin2 AF205888 G7 45 9 20% GART X54199 A10 21 4 19% Bcl-10 AF082283A8 32 6 19% RHAMM U29343 A9 57 9 16% PTEN U92436 A6 32 5 16% OGT U77413T10 78 10 13% BLM U39817 A9 170 19 11% Fas X63717 T7 30 3 10% Apaf-1AF013263 A8 43 4  9% MLH3 AF195657 A9 99 9  9% CBF M37197 A9 57 5  9%HTP1 AB024582 A9 57 5  9% RECQL L36140 A9 68 5  7% RBBP8 U72066 A9 70 5 7% MLH3 AF195657 A8 123 7  6% PTEN U92436 A6 32 2  6% CHD2 AF006514 A1019 1  5% ATRmRNA U76308 A10 77 4  5% INPPL1/DRP L24444 C7 82 4  5% CHK1AF016582 A9 21 1  5% SYCP1 X95654 A10 71 3  4% RIZ U17838 A8 83 3  4%ANG2 AF004327 A9 57 2  4% KKIAMRE/ U35146 A9 57 2  4% CDKL2   ATM U82828T7 39 1  3% CDX2 Y13709 G7 45 1  2% Axin2 AF205888 A6 45 1  2% BRCA1U14680 A8 92 2  2% Doc-1 U53445 A9 57 1  2% BRCA2 U43746 A8 119 2  2%RFC3 L07541 A10 76 1  1% Casp8AP2/ AF154415 A9 13 0  0% FLASH  ERCC5/XPG D16305 A9 13 0  0% HUMGPRKLG L03718 A9 57 0  0% DP2 U18422 A957 0  0% PMS2 U14658 A8 133 0  0% Caspase 1/ M87507 A8 42 0  0% ICE  WRN L76937 A8 11 0  0% POLA X06745 A8 83 0  0% NSEP M85234 C8 132 0  0%SHC1 U73377 G8 11 0  0% NBS1 AF058696 A7 39 0  0% BRCA2 X95152 T6 31 0 0%

TABLE 2 Coding microsatellite genes investigated in MSI-H gastriccancer. Repeat: type of nucleotide and length of repeat tract, n: numberof samples investigated, mut.: number of samples mutated, %: percentageof mutated samples gene acc.no. repeat n mut. % HPDMPK Y10936 T14 15 15100% TAF1B L39061 A11 15 13  87% PTHL3 M24350 A11 15 11  73% MACS D10522A11 15 9  60% TGFbIIR M85079 A10 227 122  54% HT001 AF113539 A11 15 8 53% MBD4/MED1 AF072250 A10 15 7  47% RIZ U17838 A9 51 23  45% Caspase 5U28015 A10 25 11  44% AIM2 AF024714 A10 15 6  40% OGT U77413 T10 15 6 40% SLC23A1 AF058319 C9 15 6  40% BAX L22473 G8 208 72  35% MSH6 U54777C8 224 76  34% ABCF1 AF027302 A10 15 5  33% FLT3LG U29874 C9 15 5  33%MSH3 J04810 A8 202 65  32% RAD50 Z75311 A9 36 10  28% PRKDC U63630 A1030 8  27% SLC4A3 U05596 C9 15 4  27% BLM U39817 A9 36 9  25% ATRmRNAU76308 A10 18 4  22% B2M AB021288 A5 28 6  21% MAC30X L19183 A10 15 3 20% RFC3 L07541 A10 15 3  20% UVRAG X99050 A10 15 3  20% AC1 D82070 T1015 3  20% IGFIIR Y00285 G8 202 36  18% Apaf-1 AF013263 A8 20 3  15%TCF-4 Y11306 A9 23 3  13% SYCP1 X95654 A10 40 4  10% Bcl-10 AF082283 A820 2  10% Fas X63717 T7 20 2  10% U79260 U79260 T14 15 1  7% GART X54199A10 15 1  7% TCF6L1 M85079 A10 15 1  7% ELAVL3 D26158 G9 15 1  7% ATMU82828 T7 36 2  6% BRCA1 U14680 A8 64 3  5% INPPL1/DRP L24444 C7 25 1 4% RIZ U17838 A8 51 1  2% CHD2 AF006514 A10 15 0  0% CHK1 AF016582 A915 0  0% BRCA2 U43746 A8 68 0  0% PMS2 U14658 A8 30 0  0% NSEP M85234 C850 0  0% NBS1 AF058696 A7 36 0  0%

The results presented in Tables 1 and 2 show, that the testedmicrosatellite regions are frequently mutated in tumor samples.

Example 2: Detection of the Expression of Frameshift Mutated mRNA fromGenes Harbouring Coding Microsatellite Regions Using PCR

Samples of colon, gastric and endometrial carcinomas are used todetermine the expression of mRNA showing frameshift mutations in codingmicrosatellite regions using PCR and subsequent sequencing of theamplified nucleic acid products.

Tumors are collected, snap frozen, and stored at −80° C. They areverified to be composed predominantly of neoplastic cells byhistopathological analysis. mRNA is isolated from tumors using Qiagenreagents (Qiagen, Hilden, Germany), and single-stranded cDNA issynthesized using Superscript II (Life Technologies, Inc.).

PCR reactions were performed in a total volume of 25 μl containing 50 ngcDNA, 2.5 μl 10× reaction buffer (Life Technologies, Karlsruhe,Germany), 1.5 mM MgCl₂, 200 μM dNTPs, 0.3 μM of each primer, and 0.5 UTaq DNA polymerase (Life Technologies) and using the followingconditions: initial denaturation at 94° C. for 4 min, followed by 35cycles of denaturation at 94° C. for 30 s, annealing at 58° C. for 45 s,and primer extension at 72° C. for 30 s. The final extension step wascarried out at 72° C. for 6 min. PCR fragments were analyzed on an ALFDNA sequencing device (Amersham Pharmacia Biotech, Freiburg, Germany)using 6.6% polyacrylamide/7 M urea gels.

The experiments described show, that in the cases of the tested genesCHD2, UVRAG, ELAVL3, TCF6L1, ABCF1, AIM2, TAF1B, MACS and HT001 mutatedcoding microsatellite regions are transcribed into mRNA.

These results indicate, that the cells harbouring mutations in codingmicrosatellite regions of the nine genes tested express neo-polypeptidesderived from these frameshift mutations.

Example 3: Stimulation of Cellular Immune Response by FrameshiftPeptides

The present experiments were performed in order to determine, whetherthe frameshift peptides arising from mutations in coding microsatelliteregions according to the present invention are suited to stimulate acellular immune response. The experiments were performed as follows:

Peptides displaying HLA-A2.1-binding motifs were selected by takingadvantage of specific computer programs [(Parker, Bednarek, & Coligan1994); bimas.dcrt.nih.gov/molbio/hla_bind/and (Rammensee et al. 1999);134.2.96.221/scripts/MHCServer.dll/home.htm].

TABLE 3 Frameshift Peptides analyzed for in vitro stimulationof a cellular immune response. All analyzed peptides(SEQ ID NOs: 75-106, as listed in Table 3 in order) are derived from (−1) mutations in microsatellites of the cognate proteins; the protein or nucleotide accession numbers are indicated within thetable; the position of the start amino acid in the proteinis indicated in the tables; the predicted bindingscores to HLA-A2.1 using computer assisted analysis; AccessionTheoretical Scores Protein Number Name Peptide Ken Parker SYFPEITHITGF-betaRII (−1) AAA61164 FSP01 ¹²⁸-SLVRLSSCV 70 23 TGF-betaRII (−1)AAA61164 FSP02 ¹³¹-RLSSCVPVA 5 19 TGF-betaRII (−1) AAA61164 FSP03¹³⁵-CVPVALMSA 1 14 HPDMPK (−1) CAA71862 FSP04 ¹³⁶-LLHSAPTPSL 36 25HPDMPK (−1) CAA71862 FSP05 ¹²⁹-FLSASHFLL 570 21 HPDMPK (−1) CAA71862FSP07 ¹²⁵-RVFFFYQHL 39 15 OGT (−1) AAB63466 FSP06 ¹²⁸-SLYKFSPFPL 397 23D070(−1) BAA11534 FSP08 ³⁵-KIFTFFFQL 1593 21 D070 (−1) BAA11534 FSP09⁶⁸-ALLPAGPLT 28 21 D070 (−1) BAA11534 FSP10 ⁶⁹-LLPAGPLTQT 29 20U79260 (−1) AAB50206 FSP11 ⁵⁹-TLSPGWSAV 118 25 U79260 (−1) AAB50206FSP12 ⁸³-ILLPQPPEWL 362 26 Sec63 (−1) AAC83375 FSP13 ⁵⁵¹-RQMESLGMKL 3315 MAC30X (−1) AAA16188 FSP14 ¹⁹⁸-VEMPTGWLL 20 14 MAC30X (−1) AAA16188FSP15 ¹⁹⁸-VEMPTGWLLV 14 15 FLT3L (−1) U29874 FSP16 ¹¹³-FQPPPAVFA 13 10MSH-3 (−1) AAB47281 FSP17 ³⁸⁹-ALWECSLPQA 389 24 MSH-3 (−1) AAB47281FSP18 ³⁸⁶-FLLALWECSL 364 25 MSH-3 (−1) AAB47281 FSP19 ³⁸⁷-LLALWECSL 3626 MSH-3 (−1) AAB47281 FSP20 ³⁹⁴-SLPQARLCL 21 23 MSH-3 (−1) AAB47281FSP21 ⁴⁰²-LIVSRTLLL 5 23 MSH-3 (−1) AAB47281 FSP22 ⁴⁰¹-CLIVSRTLL 21 22MSH-3 (−1) AAB47281 FSP23 ⁴⁰³-IVSRTLLLV 24 21 MSH-3 (−1) AAB47281 FSP24³⁸²-KRATFLLAL 0.1 20 Caspase-5 (−1) U28015 FSP25 ⁶¹-KMFFMVFLI 1301 20Caspase-5 (−1) U28015 FSP26 ⁶⁷-FLIIWQNTM 22.85 21 TAF-1b (−1) L39061FSP27 ¹⁰⁸-GMCVKVSSI 17 24 HT001 (−1) NP 054784 FSP30 ²⁸¹-VLRTEGEPL n.d.21 MSH-3 (−1) AAB47281 FSP31 ⁴⁰²-LIVSRTLLLV 37 25 MSH-3 (−1) AAB47281FSP32 ³⁹⁴-SLPQARLCLI 24 24 MSH-3 (−1) AAB47281 FSP33 ⁴⁰¹-CLIVSRTLLL 2123 MSH-3 (−1) AAB47281 FSP34 ³⁹⁹-RLCLIVSRTL 4 22

Peptides were purchased from the peptide synthesis unit of the DKFZ.Stock solutions (10 mg/ml in DMSO) were stored at −70° C. and diluted to1 mg/ml in PBS before use. T2 cells were pulsed with 50 μg/ml peptideand 5 μg/ml ß2-microglobulin (Sigma; Deisenhofen, Germany) overnight at37° C. The expression of HLA-A2.1 was then analyzed by flow cytometryusing mAb BB7.2 followed by incubation with FITC-conjugated (ab′)2 goatanti-mouse Ig (Vonderheide et al. 1999).

Peripheral blood was obtained from a healthy HLA-A2.1+ donor andcollected in heparinized tubes. PBMNC were isolated by Ficoll-densitygradient centrifugation. Whole CD3+ T-cells were isolated from PBMNC bymagnetic depletion of non-T-cells using the MACS Pan T-cell IsolationKit (Miltenyi; Bergisch Gladbach, Germany) according to manufacturer'sinstructions. Preparations contained at least 97% of CD3+ cells asassessed by immunophenotypic analysis.

HLA-A2.1-restricted peptides were FSP27 and FSP29 from a (−1) in theTAF1B-gene; FSP30 was derived from a (−1) mutation in the HT001 gene.

CD40 Bs of a HLA-A2.1+ donor were incubated with peptide (10 μg/ml) andhuman ß2-microglobulin (3 μg/ml; Sigma) in serum-free Iscov's DMEMmedium for one hour at room temperature, washed twice to remove excessof peptide, were irradiated (30Gy) and added to purified CD3+ autologousT-cells (>97% CD3+) at a ratio of 4:1 (T:CD40 Bs) in Iscov's MEMcontaining 10% human AB-serum, supplements (1:100) and hIL-7 (10 ng/ml,R&D). Cells were plated at a density of 2×106 T-cells/well in 1 ml ofmedium. After three days in culture they were fed with 1 ml completemedium. For restimulation of T-cells, this was repeated weekly. IL-2 wasfirst given at day 21 (10 IU/ml, R&D), also at day 24 and from day 28 ononly hIL-2 was used instead of hIL7.

ELISpot assays were performed as described elsewhere (Meyer et al.1998). Briefly, nitrocellulose-96-well plates (Multiscreen; Millipore,Bedford, USA) were covered with mouse anti-human IFN-g monoclonalantibody (Mabtech, Sweden) and blocked with serum containing medium.Varying numbers of effector cells were plated in triplicates with3.5×104 peptide-loaded T2 cells per well as targets. After incubationfor 18 h, plates were washed, incubated with biotinylated rabbitanti-human IFN-g second antibody, washed again, incubated withstreptavidin coupled alkaline phosphatase, followed by a final wash.Spots were detected by incubation with NBT/BCIP (Sigma) for 45 min,reaction was stopped with water, after drying spots were counted usingthe KS-ELISpot reader (Zeiss Kontron; Gottingen, Germany).

The analysis shows, that the used peptides are suited to raise an immuneresponse. Peptides arising from frameshift mutations thus may be used toraise immune response for example in the course of vaccinationsaccording to the present invention.

Example 4: Screening for Antibodies Directed Against Frameshift Peptidesin Patient Samples

Serum of 25 patients with diagnosed colorectal carcinomas was tested forthe presence of antibodies to a set of frameshift peptides arising fromframeshift mutations in coding microsatellite regions of the followinggenes: CHD2, UVRAG, ELAVL3, TCF6L1, ABCF1, AIM2, TAF1B, MACS and HT001.As a control the serum of 50 normal individuals was tested.

For the test synthetic peptides representing immunogenic portions of allrelevant frameshift peptides (see FIGS. 2-1 to 2-8) arising from therespective genes were spotted onto nylon membranes. The nylon membraneswere subsequently incubated for one hour in phosphate-buffered saline(PBS) with 5% milk powder for blocking unspecific membrane binding.After washing the membranes 3× with PBS, the membranes were incubatedwith the test and control sera. The sera were diluted 1:1.000 inPBS/0,5% milk powder and incubated overnight with gentle shaking.Subsequently the sera were removed, and membranes were washed threetimes in PBS before they were incubated with a polyclonal alkalinephosphatase conjugated goat anti-human IgG antibody for one hour.Thereafter, the membranes were washed repeatedly with PBS/0,05% TWEEN20before staining reaction was developed using nitroblue tetrazoliumchloride and bromochoro-indoyl-phosphate (SigmaAldrich) in Tris-bufferedsaline (TBS). Binding of human antibodies specific for individualframeshift polypeptides thus was made visible by color-deposit on therespective membrane.

The results show, that in all samples of tumor patients antibodiesdirected against at least one peptide arising from frameshift mutationswere present.

This illustrates, that the method according to the present invention maybe used for diagnosis of diseases associated with frameshift mutationsin coding regions of genes.

Example 5: In Vitro Stimulation of Cellular Immune Response byFrameshift Peptides

The present experiments were performed in order to determine, whetherthe frameshift peptides arising from mutations in coding microsatelliteregions according to the present invention are suited to stimulate acellular immune response. The experiments were performed as follows:

Peptides displaying HLA-A2.1-binding motifs were selected by takingadvantage of specific computer programs [(Parker, Bednarek, & Coligan1994); bimas.dcrt.nih.gov/molbio/hla_bind/and (Rammensee et al. 1999);134.2.96.221/scripts/MHCServer.dll/home.htm]. Peptides were purchasedfrom the peptide synthesis unit of the DKFZ. Stock solutions (10 mg/mlin DMSO) were stored at −70° C. and diluted to 1 mg/ml in PBS beforeuse. T2 cells were pulsed with 50 μg/ml peptide and 5 μg/mlß2-microglobulin (Sigma; Deisenhofen, Germany) overnight at 37° C. Theexpression of HLA-A2.1 was then analyzed by flow cytometry using mAbBB7.2 followed by incubation with FITC-conjugated (ab′)2 goat anti-mouseIg (Vonderheide et al. 1999).

Peripheral blood was obtained from a healthy HLA-A2.1+ donor andcollected in heparinized tubes. PBMNC were isolated by Ficoll-densitygradient centrifugation. Whole CD3+ T-cells were isolated from PBMNC bymagnetic depletion of non-T-cells using the MACS Pan T-Cell IsolationKit (Miltenyi; Bergisch Gladbach, Germany) according to manufacturer'sinstructions. Preparations contained at least 97% of CD3+ cells asassessed by immunophenotypic analysis.

CD40 Bs of a HLA-A2.1+ donor were incubated with peptide (10 μg/ml) andhuman ß2-microglobulin (3 μg/ml; Sigma) in serum-free Iscov's DMEMmedium for one hour at room temperature, washed twice to remove excessof peptide, were irradiated (30Gy) and added to purified CD3+ autologousT-cells (>97% CD3+) at a ratio of 4:1 (T:CD40 Bs) in Iscov's MEMcontaining 10% human AB-serum, supplements (1:100) and hIL-7 (10 ng/ml,R&D). Cells were plated at a density of 2×10⁶ T-cells/well in 1 ml ofmedium. After three days in culture they were fed with 1 ml completemedium. For restimulation of T-cells, this was repeated weekly. IL-2 wasfirst given at day 21 (10 IU/ml, R&D), also at day 24 and from day 28 ononly hIL-2 was used instead of hIL7.

ELISpot assays were performed as described elsewhere (Meyer et al.1998). Briefly, nitrocellulose-96-well plates (Multiscreen; Millipore,Bedford, USA) were covered with mouse anti-human IFN-g monoclonalantibody (Mabtech, Sweden) and blocked with serum containing medium.Varying numbers of effector cells were plated in triplicates with3.5×10⁴ peptide-loaded T2 cells per well as targets. After incubationfor 18 h, plates were washed, incubated with biotinylated rabbitanti-human IFN-g second antibody, washed again, incubated withstreptavidin coupled alkaline phosphatase, followed by a final wash.Spots were detected by incubation with NBT/BCIP (Sigma) for 45 min,reaction was stopped with water, after drying spots were counted usingthe KS-ELISpot reader (Zeiss Kontron; Gottingen, Germany).

These procedures were performed for peptides derived from mutations inthe coding regions of the following genes: TGFβRII, OGT, U79260, CASP 5,MSH 3, HPDMPK, HT001, TAF1B, D070, MAC30X, FLT3L and SEC63. Peptides arelisted in FIGS. 2-1 to 2-8. Results for ELIspot analysis is shown inFIGS. 1-1 to 1-2.

The analysis shows, that the used peptides are suited to raise an immuneresponse. Peptides arising from frameshift mutations thus may be used toraise immune response for example in the course of vaccinationsaccording to the present invention.

Example 6: Cytotoxicity Assay Directed Against Cells DisplayingFrameshift Peptides

CTL bulk cultures and/or CTL clones obtained according to the methoddescribed in Example 1 were tested for their cytotoxicity as follows:

Due to the limited amount of cell material clones were in someexperiments pooled for the determination of the cytotoxicity.

To obtain cells presenting frameshift peptides on the one hand differentMSI+ cell lines endogenously expressing mutated mRNA of the respectiveframeshift peptides of TGWU, OGT, U79260, CASP 5, MSH 3, HPDMPK, HT001,TAF1B, D070, MAC30X, FLT3L and SEC63 either expressed HLA-A2.1endogenously or were stably transfected with HLA-A2.1 on the other handseveral MSI− cell lines expressing HLA-A2.1 were transfected with themutated full-length cDNA of the respective frameshift peptides (of TGWU,OGT, U79260, CASP 5, MSH 3, HPDMPK, HT001, TAF1B, D070, MAC30X, FLT3Land SEC63). After selection and expansion of the transfected cell linesfor each respective frameshift peptide at least two stably transfectedsub-cell lines were available. These cell lines were used incytotoxicity assays, wherein negative controls were the respectiveuntransfected MSI+ HLA-A2.1 negative and/or the MSI−, HLA-A2.1 positivecell lines.

It could be shown, that the transfected cell lines were lysed by thebulk cultures and/or pooled clones of CTLs. The reactivity was tested atdifferent target cell to effector cell ratios. In average around 20%-30%of the target cells were lysed in the assays. The control cells werealways lysed at a significantly lower percentage.

In FIGS. 3 and 4 results for the frameshift peptides FSP02 (TGFβRII(−1))and FSP06 (OGT(−1)) respectively are shown. These results shall berepresentative for the results related to the other frameshift peptides,which rendered similar rates of lysis.

The experiments show, that frameshift peptides may generate immuneresponse. The frameshift peptides may furthermore be applied for thedetection of the presence of cytotoxic T-cells directed against aparticular frameshift peptide.

Example 7: Screening for Antibodies Directed Against Frameshift Peptidesin Patient Samples

Serum of 25 patients with diagnosed colorectal carcinomas was tested forthe presence of antibodies to a set of frameshift peptides arising fromframeshift mutations in coding microsatellite regions of the followinggenes: TGWU, U79260, CASP 5, HT001, PTHL3, MACS, TCF4, TAF1B, AC1, AIM2,SLC23A1, ABCF1, HSPC259, BAX, TCF6L1, FTL3L, OGT, ELAVL3, MAC30X,MAC30X, SLC4A3, PRKDC, UVRAG, MSH3 and SEC63. As a control the serum of50 normal individuals was tested.

For the test synthetic peptides (20 to 40 mers, partly overlapping)representing immunogenic portions of all relevant frameshift peptides(see FIGS. 2-1 to 2-8) arising from the respective genes were spottedonto nylon membranes. The nylon membranes were subsequently incubatedfor one hour in phosphate-buffered saline (PBS) with 5% milk powder forblocking unspecific membrane binding. After washing the membranes 3×with PBS, the membranes were incubated with the test and control sera.The sera were diluted 1:1.000 in PBS/0,5% milk powder and incubatedovernight with gentle shaking. Subsequently the sera were removed, andmembranes were washed three times in PBS before they were incubated witha polyclonal alkaline phosphatase conjugated goat anti-human IgGantibody for one hour. Thereafter, the membranes were washed repeatedlywith PBS/0,05% TWEEN20 before staining reaction was developed usingnitroblue tetrazolium chloride and bromochloro-indoyl-phosphate(SigmaAldrich) in Tris-buffered saline (TBS). Binding of humanantibodies specific for individual frameshift polypeptides thus was madevisible by color-deposit on the respective membrane.

The results show, that in all samples of tumor patients antibodiesdirected against at least one peptide arising from frameshift mutationswere present.

This illustrates, that the method according to the present invention maybe used for diagnosis of diseases associated with frameshift mutationsin coding regions of genes.

What is claimed is:
 1. A method comprising measuring the presence orabsence or level of antibodies or antigen-recognizing cells directedagainst a frameshift polypeptide in a biological sample from a cancerpatient selected from the group consisting of a lung cancer patient,liver cancer patient, renal cancer patient, melanoma patient, cancer ofthe brain patient, head and neck cancer patient, leukemia patient,lymphoma patient, prostate cancer patient, breast cancer patient, ovarycancer patient, endometrial cancer patient, lung cancer patient, andbladder cancer patient, wherein the frameshift polypeptide is derivedfrom a frameshift mutation in the A11 repeats in a coding microsatelliteregion of a gene selected from the group consisting of TAF1B, MACS, andHT001.
 2. The method of claim 1, wherein the frameshift polypeptide isderived from the A11 repeat of the TAF1B gene.
 3. The method of claim 1,wherein the frameshift polypeptide is derived from the A11 repeat of theHT001 gene.
 4. The method of claim 1 wherein the frameshift polypeptideis derived from the A11 repeat of the MACS gene.
 5. The method of claim1, which measures the presence or absence and/or level of theantibodies.
 6. The method of claim 1, which measures the presence orabsence and/or level of the antigen-recognizing cells.
 7. The method ofclaim 1, wherein colorectal cancer patient is a patient in which thepresence or absence and/or level of antibodies or antigen-recognizingcells against the one or more frameshift polypeptides has not previouslybeen detected.
 8. The method of claim 1, wherein the presence or absenceand/or level of a plurality of antibodies or antigen-recognizing cellsis measured, each of the plurality directed against one of a set offrameshift polypeptides, the set of frameshift polypeptides comprisingat least one of the frameshift polypeptides derived from the frameshiftmutation in the A11 repeats in the coding microsatellite region of thegene selected from the group consisting of TAF1B, MACS, and HT001. 9.The method of claim 8, wherein the set of frameshift polypeptidesfurther comprises one or more frameshift polypeptides derived from aframeshift mutation in the A11 repeats in a coding microsatellite regionof a gene selected from the group consisting of UVRAG, ELAVL3, TCF6L1,ABCF1, AIM2, and CH D2.
 10. The method of claim 8, wherein the set offrameshift polypeptides comprises at least 3 frameshift polypeptides.11. The method of claim 8, wherein the set of frameshift polypeptidescomprises at least 4 frameshift polypeptides.
 12. The method of claim 8,wherein the set of frameshift polypeptides comprises at least 5frameshift polypeptides.
 13. The method of claim 8, wherein the set offrameshift polypeptides comprises up to 30 frameshift polypeptides. 14.The method of claim 8, wherein the set of frameshift polypeptides isselected from the group consisting of: Set 1, consisting of HT001,U79260, and MACS; Set 2, consisting of HT001, TAF 1B, and MACS; Set 3,consisting of HT001, TGFB2R, and MACS; Set 4, consisting of HT001,U79260, and TGFB2R; Set 5, consisting of HT001, U79260, and TAF1B; Set6, consisting of HT001, TGFB2R, and TAF1B; Set 7, consisting of HT001,U79260, TGFB2R, and MACS; Set 8, consisting of HT001, U79260, TGFB2R,and AC1; Set 9, consisting of HT001, U79260, TGFB2R, and TAF1B; Set 10,consisting of HT001, TGFB2R, MACS, and CASP5; Set 11, consisting ofHT001, U79260, MACS, and CASP5; Set 12, consisting of HT001, U79260;MACS, and AC1; Set 13, consisting of HT001, TGFB2R, TAF1B, and CASP5;Set 14, consisting of HT001, U79260, MACS, and OGT; Set 16, consistingof HT001, U79260, TGFB2R, MACS, and AC1; Set 17, consisting of HT001,U79260, TGFB2R, TAF1B, and MACS; Set 18, consisting of HT001, U79260,TGFB2R, TAF1B, and AC1; Set 19, consisting of HT001, U79260, TGFB2R,MACS, and AIM2; Set 20, consisting of HT001, U79260, TGFB2R, TAF1B, andAIM2; Set 21, consisting of U79260, TGFB2R, TAF1B, AC1, and CASP5; a setconsisting of HT001, U79260, TGFB2R, AC1, and CASP5; Set 22, consistingof U79260, TGFB2R, MACS, AC1, and CASP5; Set 23, consisting of HT001,U79260, TAF1B, MACS, and AC1; Set 24, consisting of HT001, U79260,TAF1B, MACS, and CASP5; a set consisting of HT001, U79260, MACS, AC1,and OGT; Set 25, consisting of HT00, U79260, MACS, MSH3, and OGT; Set26, consisting of HT001, U79260, TGFB2R, MACS, and OGT; Set 27,consisting of HT001, TGFB2R, TAF1B, AC1, and CASP5; and Set 28,consisting of HT001, U79260, TGFB2R, AC1, and AIM2.